Method for culturing microorganisms in prefilled flexible containers

ABSTRACT

The invention is a method for using flexible packaging in connection with incubating a sample in a liquid culture medium. A flexible, gusseted bag is first presterilized by irradiation. Then, the bag is prefilled with a presterilized liquid culture medium. The bag is self-supporting and takes on the characteristics of a rigid container when filled. Prefilling and sterilizing it enables it to be shipped to a laboratory. The bag is easy to open and reseal for introduction of a culture sample at the laboratory. After introduction, the sample is incubated in the bag.

RELATED APPLICATION

This application is a continuation of application Ser. No. 09/185,375,filed Nov. 3, 1998, now U.S. Pat. No. 6,146,875, which is acontinuation-in-part (CIP) of application Ser. No. 08/850,837, filed May2, 1997, now abandoned.

TECHNICAL FIELD

This invention relates to microbiology, more particularly, it relates toa method for prefilling, sterilizing, and shipping culture media inflexible packaging, and then introducing samples in the prefilledpackaging for culturing microorganisms.

BACKGROUND INFORMATION

Microbiologists incubate samples in sterile liquid culture media todetect and perform tests for pathogenic microorganisms. Salmonella,Listeria, Campylobacter and E. coli are some of the typicalmicroorganisms subject to culturing in this manner. Similar kinds oftests are conducted to detect the presence of microorganisms in samplesnormally expected to be sterile, such as blood, spinal fluid, medicaldevices, and a wide variety of industrial materials.

Historically, microbiologists prepare sterile liquid culture solutionsand buffered diluent solutions (collectively “culture media”) in glassor plastic bottles (“rigid containers”). Although rigid containers canbe reused, the preparation of culture media in these containers isexpensive, labor intensive, and subject to error.

A laboratory that prepares its own culture media typically undertakes aseries of steps. First, the container is washed and rinsed to remove anyresidual substances or chemicals that may inhibit the growth ofmicroorganisms. Then a measured amount of purified water and a powderedculture mix are placed in the container. The water is heated to dissolvethe mix in the water and Ph adjustments are made, as necessary. This isfollowed by sterilizing the container and its contents in an autoclave.The test sample is then introduced into the culture medium aftersterilization.

Because the foregoing procedure is labor intensive, many laboratoriesnow prefer to purchase prefilled and presterilized rigid containers,rather than conduct mixing and sterilization themselves. This leaves thelaboratory with the task of merely introducing test samples into thecontainers after they arrive.

Regardless of whether a lab makes and sterilizes its own culture mediaor purchases prefilled and presterilized containers, rigid containershave been the containers of choice. They can easily withstand theautoclave conditions (121° C., 15 p.s.i., 100% steam) necessary tosterilize liquid culture media.

Glass is particularly advantageous as a container because it allowsvisual inspection of the culture media before and after a sample isadded. Because it is a rigid container, it can be easily moved fromplace to place. It rests easily on any flat surface and does not requiresupporting racks or similar strictures.

In the prefilled situation, the impermeability of glass extends theshelf life of the culture media by preventing evaporative loss. Whileplastic is used in many instances because it weighs less than glass andis relatively resistant to breakage, many types of plastics cannotwithstand autoclave conditions. The types of plastics which canwithstand autoclave conditions are expensive, have reduced clarity, andtend to distort or break after repeated autoclave exposure.

In general, rigid containers are expensive to make (the cost of a capfor capped containers can be as much as 25% of the total cost of thecontainer), heavy to ship, subject to breakage, and contribute to totalwaste disposal. The breakage and weight problems associated with rigidcontainers are particularly disadvantageous when they are prefilled andpresterilized at one geographic location and then shipped to anotherlocation for use.

A further problem with rigid containers is that they do not allow a userto easily mix a sample into the culture media. When mixing is required,the container must be shaken to adequately distribute the sample in themedia. If shaking will not work because of sample type, the contents ofthe container must be transferred to a blender bag which is then placedin a machine having reciprocating paddles that pulverize and mix thesample with the culture media. After mixing, the sample and media mustbe returned to the rigid container for incubation.

As will become apparent, the present invention solves the above problemsand provides a more convenient, less expensive, and better way toculture samples.

SUMMARY OF THE INVENTION

The invention is a method for producing a liquid culture medium such as,for example, culture broth or a diluent solution. Ultimately, theculture medium is to be used for incubating or culturing a sample.

The invention incorporates the use of a gusseted plastic bag. Fordefinitional purposes, a gusseted bag is one having a gusseted basewhich allows the bag to stand alone without any external support. In thepreferred embodiment, the gusseted bag is comprised of a pair of thinfilm plastic sheets that lay one on top of the other when the bag is notfilled with a culture medium. The lower portion or region of the sheetsare connected together and closed to form the gusseted base. The sideedges of the sheets are bonded directly to each other without the use ofa gusset. The upper portion or region of the sheets may be spread apartin order to fill the bag.

In accordance with one embodiment of the invention, the liquid culturemedium is sterilized before it is placed in a presterilized bag.Presterilization of the liquid culture medium is done by placing themedium in an autoclave and subjecting it to sterilization temperaturesand pressures. According to what is presently believed to be thepreferred embodiment, the bag is presterilized using a separateprocedure of irradiation to kill any microorganisms.

The presterilized bag is then filled with the presterilized culturemedium in a clean room. Since the bag is gusseted and self-supporting,it stands up by itself when filled and can be moved from place to placelike a rigid container.

The prefilled and presterilized bag is then shipped to a lab forincubating a sample (sometimes called “culture sample”). The culturesample is added to the culture medium by opening the bag andsubsequently sealing it. Preferably, the bag is designed to have aresealable opening in the form of a “ziplock” closure, although otherways of sealing the bag may work just as well. After sealing, the sampleis incubated in accordance with known incubation procedures, accordingto sample type.

An alternative method of resealing the bag is to use a closure wireinstead of a “zip lock” closure. The closure wire is connected to theupper portion of one of the sheets, and the closure wire has a lengththat exceeds the width of the bag. After the culture medium is added tothe bag, the upper portions of the pair of sheets, above the wireclosure, are bonded together to form an airtight seal. When the bagarrives at the laboratory for use, the upper portion of the bag isspread open to break the airtight seal, allowing a culture sample to beinserted into the bag. Afterwards, to reseal the bag, the upper portionof the bag is folded or rolled about the closure wire, and then the endsof the closure wire are wrapped around the rolled upper portion, toprevent it from unrolling.

In an alternative embodiment, the bag and medium are sterilized in thesame step by radiation treatment. In this embodiment, a non-sterileculture broth is added to a non-sterile bag and then subjected toradiation treatment by gamma rays or electrons to render the productsterile. This alternative will be further described below.

An advantage of the invention is that it works particularly well insituations where the sample is not easily dissolved in the culturemedium. In some cases, in order to obtain a good distribution of thesample, it is necessary to physically stir or beat the mixture. When theabove process is used, the solution of medium and sample can be quicklypulverized or kneaded through the walls of the flexible bag, as needed,without transferring bag contents from one container to another. Thisreduces the risk of introducing unwanted contaminants. As mentionedabove, container to container transfer is a drawback in this situationif rigid containers are used.

A second advantage of the invention is that use of a clear, gusseted bagallows visual inspection of the culture medium and sample at any timeduring the culturing or test process. For example, a user can easilydetermine whether the contents of a prefilled and sterilized bag havebeen contaminated at any point in time during shipment or storage.Typically, a prefilled and presterilized bag will be quarantined for aperiod of time as a manufacturer's test for sterility prior to supplyingit to a laboratory. Quarantine will reveal contamination by visualinspection. If the bag passes quarantine, then it is deliverable to thelaboratory as a “ready-to-use” unit for incubation purposes. Similarly,after the sample is placed in the bag and incubated, the user canvisually inspect it for microorganism growth.

A third and most important advantage of the invention is that itsignificantly reduces laboratory costs. There is now a significantmarket relating to supplying labs with prefilled and presterilizedculture media in ready-to-use, rigid containers. However, there is nomechanism in place for easy recycling of the containers back to thesupplier for reuse. Consequently, the containers tend to be used onceand then disposed of by the lab. Not only does this create unnecessarywork, but the incremental cost of the container alone is significantrelative to what is supplied to the laboratory. Replacing rigidcontainers with gusseted bags or pouches retains all the existingadvantages of rigid containers and provides additional advantagesrelating to significantly lower costs per container (a cost savings ofapproximately 20-40% per container) and a reduction in total mass andvolume of waste material.

Having briefly summarized the invention, it will become betterunderstood after review of the following description, which is to betaken in conjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference numerals and letters refer to like partsthroughout the various views unless specifically indicated otherwise,and wherein:

FIG. 1 is a pictorial view of a gusseted pouch constructed in accordancewith the invention;

FIG. 2 is the first in a series of two flow charts outlining the varioussteps of the invention;

FIG. 3 is the second in a series of two flow charts outlining thevarious steps of the invention;

FIG. 4 is a pictorial view of a plurality of gusseted pouches about tobe placed in a shipping pouch;

FIG. 5 is a pictorial view of an incubator;

FIG. 6 is a side cross-sectional view of the incubator shown in FIG. 5;

FIG. 7 is a view like FIG. 1 but shows an alternative embodiment of thegusseted pouch;

FIG. 8 is a view like FIGS. 1 and 7, but shows a closure wire for thegusseted pouch.

FIG. 9 is a pictorial view like FIG. 8 but shows a pipette sock insidethe pouch;

FIG. 10 is an enlarged, fragmented view of the pipette sock shown inFIG. 9; and

FIG. 11 is a pictorial view of the pipette sock and shows the taperedend of a pipette about the be inserted into an upper opening in thesock.

BEST MODE FOR CARRYING OUT THE INVENTION

Referring to the drawings, and first to FIG. 1, shown generally at 10 isa gusseted bag or pouch that is designed for use in conjunction with theinvention. The pouch 10 has a gusseted base 12 which enables it to standalone in vertical orientation when it is filled with a liquid medium 14.A person skilled in the art would be familiar as to how the bag is made.However, for the purpose of using it in the method described below, thepouch 10 must be made of a material that is resistant to puncturing,substantially rigid to stand alone in a vertical position, suitablytransparent to allow visual inspection of the contents, and has a longshelf life. Preferably, the pouch 10 is comprised of two sheets of thinfilm, transparent plastic sheets 22 and 24, that lay one on top of theother when the pouch 10 is not filled. The lower portion or region 28 ofthe sheets 22 and 24 are connected together and closed to form thegusseted base 12. The side edges of the sheets are bonded directly toeach other without a gusset, in the manner shown in FIGS. 1 and 8, suchthat the pouch 10 is a “two-sided” pouch and will lay flat, one sheet ontop of the other. The upper portion of the pouch 10, near the edges 18and 20 of the sheets 22 and 24, arc bonded together in a manner thatwill be described later below.

Various types of suitable bag materials arc available from a number ofsources. The pouch 10 should be made of laminated plastic films.Suitable materials are available from Riley & Geehr in Evanston, Ill.,identified by the following product numbers: DF#4400 (Product No. fromRiley & Geehr) 48 gauge (0.00048 inches) PET/Adhesive/2.5 gauge LLDPE(overall thickness is 3.1 mil); DF#512 (Product No. from Riley & Geehr),60 gauge Nylon/Adhesive/3.5 gauge LLDPE (overall thickness is 4.1 mil);DF#300 (Product No. from Riley & Geehr), 72 gauge NylonPVDC/Adhesive/3.5gauge LLDPE (overall thickness is 4.35 mil).

The pouch 10 is sealed by a “zip-lock” closure, indicated generally at16. A person skilled in the art would be familiar with the way this typeof closure works. In the alternative, the pouch 10 could be sealed byrolling its upper edges 18, 20 together and clipping the rolled portionwith a spring clamp. This is not shown in the drawings, however.

An alternative method of sealing the pouch 10 after the culture medium14 has been added is shown in FIG. 8, and involves simply bonding theupper edges 18, 20 together by heat sealing or the like to form anairtight seal 80. The fashion in which the edges 18, 20 are bondedtogether to form the seal 80 is known by those skilled in the art. It isnot crucial that the seal 80 be located exactly at the edges 18, 20 ofthe pouch 10—the seal 80 need only be on the upper portion of the pouch10. What is important is that the seal 80 is airtight.

FIG. 8 also shows notches 82, 84 cut into the edges of the pouch 10,just below the seal 80, to facilitate the tearing open of the bag foradding a culture sample. A flexible closure wire 86 is attached to oneof the sheets 22 or 24, below the notches 82, 84. The length of theclosure wire 86 exceeds the width of the pouch 10, such that both ends88, 90 of the closure wire 86 extend past the edges of the pouch 10.

In an alternative embodiment, a pipette sock 90 is attached to one ofthe inner walls of the pouch 10 (see FIG. 9). The pipette sock 90 ismade of a mesh-type cloth or similar material that will enable themedium and microorganisms in the medium to pass through its walls, butat the same time, serves as a filter for particulate matter. Forexample, in use, a pipette 92 (see FIG. 11) may be inserted into thesock (after the pouch 10 is open, of course) and the medium may be drawnfrom inside the pipette sock 90. The sock 90 filters the medium.

An upper end 94 of the sock is open for receiving the pipette 92. Thelower end is closed. The sock 90 is sealed at 96 to the pouch 10 (seeFIG. 10). Any material suitable for use as a filtration medium in thecontext just described may be used as a pipette sock 90.

When filled, the liquid culture medium 14 applies outward pressure toopposite sides 22, 24 of the pouch 10. This forces sides 22, 24 awayfrom each other. At the base 12 of the pouch 10, the bottom or gusset 26unfolds. The gusset 26 both defines the floor of a liquid holding vesseland constrains the outward movement of sides 22, 24. The lower sideedges 28 are stiffened by the opening of the gusset 26 and define astable base that enables the pouch 10 to rest on any flat surface 30 inthe vertical arrangement shown in FIG. 1. When the pouch 10 is closed,it is easy to move from place to place and can be placed on shelves orin boxes along with other pouches of the same construction.

In accordance with what is considered to be the invention here, anddirecting attention now to FIGS. 2 and 3, the pouch 10 is firststerilized by irradiation. Irradiation is a conventional procedure thatwould be familiar to the skilled person. Irradiation of the pouch 10 isnecessary because it is unlikely the materials used to make the pouchcan withstand autoclave conditions.

In a parallel step (see 32 in FIG. 1), the culture medium is separatelysterilized in an autoclave. Typical autoclave conditions for sterilizingthe culture medium involves subjecting the medium to a temperature of121° C., a pressure of 15 p.s.i., and 100% steam.

The culture medium may consist of any one of a number of conventionalformulations used for culturing samples, such as Nutrient Broth, SoybeanCasein Digest Broth, Thioglycollate Broth, and Brain Heart InfusionBroth. It is typically known as a culture “broth,” which would beunderstood by a person skilled in the art.

Sterilization of the pouch 10 and culture medium will occur in a cleanroom under stringent conditions. As indicated at 31, the sterilizedmedium is placed in the pouch 10, as indicated at 33. Depending onmarket demand, any number of pouches will be filled with various typesof culture media. The pouches are sealed (see 34) and the process isrepeated as many times as needed to supply market demand. This isschematically indicated at 36 in FIG. 2.

In the summary of the invention, an alternative embodiment was describedinvolving subjecting a non-sterile culture broth to a non-sterile bagand then subjecting both items to a radiation treatment as a singleunit. It is believed gamma rays or electron radiation will render theunit sterile in this situation if applied properly.

If the unit is processed in this way, it is critical that anymicroorganisms not be allowed to grow to significant levels prior to theradiation treatment. If the growth of the contaminants is not restrictedbeforehand, then the radiation dosage may not be adequate to completelykill these microorganisms. Also, if the contaminants grow to high levelsprior to sterilization, even though the radiation treatment maysubsequently kill them all, their presterilization use of criticalnutrients (e.g., proteins, amino acids, vitamins, sugars, oxygen) in themedium could result in an inability of the medium to subsequentlysupport the growth of microorganisms when subsequently used forculturing. Moreover, excessive growth of these organisms prior tosterilization may result in the creation and accumulation of toxic wasteproducts that will not be removed by sterilization, but may neverthelessrestrict or prevent the growth of microorganisms during culturing.Control of the presterilization growth of the contaminants isaccomplished by sterilizing within a short period of time after filling(e.g., 48 hours) or by refrigerating to restrict the growth of thecontaminant microorganisms.

Radiation dosage required for sterilization, regardless of whichembodiment is used, needs to be determined by experience and may varydepending on pouch material and type of culture medium. Based on currentdata, a gamma radiation dose of 2.5 Mrads should be sufficient to killmicroorganisms with an adequate safety factor.

It has recently been found that this alternative embodiment may now bepreferred. Gamma radiation in the range of 15 to 30 kGy is currently inuse.

After prefilling and sterilization, the pouches are quarantined for aperiod of time. Quarantine will reveal any contamination in the pouchesand enable culling out unusable pouch and media units prior tolaboratory shipment (see 38 in FIG. 2). Quarantine may not be necessaryfor units that are sterilized together by gamma or electron beamtreatment.

When a laboratory places an order, one or more prefilled andpresterilized pouch units are placed in a shipping pouch and sent to thelaboratory (see 40 and 42, respectively). Directing attention briefly toFIG. 4, each prefilled pouch 10A, B, C is placed within a foil shippingpouch 44. The shipping pouch 44 is preferably made of DF#505 (ProductNo. from Riley & Geehr). This material is 48 gauge chemically-treatedpolyester (provides rigidity). It has a sealing layer of low densitypolyethylene and an aluminum foil barrier. Like the pouch 10, it has azip-lock closure 46 which provides a double-seal against leakage ormoisture escape from each pouch 10A-C placed in the shipping pouch 44.In FIG. 4, pouches 10A, C are shown lying on their sides on a flatsurface 46. Pouch B is standing vertically in the same way as shown inFIG. 1.

Referring now to FIG. 3, as shown at 50, the laboratory receives ashipping pouch 44, opens it, and is able to visually inspect pouchcontents immediately. Oftentimes, a visual inspection will revealmicroorganism growth in the pouch which means that its contents somehowbecame contaminated despite the earlier quarantine step. If the visualinspection indicates no abnormalities, then the pouch 10 is used toculture a sample.

Culturing is accomplished by opening pouch 10, introducing a culturesample, and thereafter sealing the pouch (see 52). In some cases, it isnecessary to mix pouch contents. This may be a function of the type ofsample being cultured. In any event, if mixing is required, it is asimple matter to knead the pouch contents through the flexible walls ofthe pouch 10 (see 54). In some instances, a mesh bag is placed insidethe gusseted bag (see FIG. 7). The purpose of this mesh bag is to filterparticles so that if a serological pipette is used to remove bagcontents, the pipette will not become clogged. After thorough mixing,the pouch and its contents are placed in an incubator (see 56).

A “zip-lock” closure for the pouch 10, such as that shown in FIGS. 1 and7, allows for convenient opening and resealing of the pouch when addinga culture sample to the medium 14. An alternate and probably preferredmethod involves use of the closure wire 86, previously described aboveand shown in FIG. 8. To add a sample, the pouch 10 is cut open withscissors or torn open by applying the requisite force at one of thenotches 82 or 84 to tear and spread apart the sheets 22 and 24. Thesample can be added to the culture medium 14 after the opening has beentorn to a sufficient size. To reseal the pouch 10 after the sample hasbeen added, the top edges 18, 20 of the pouch, including the openingthat was created, are flattened together and then rolled about theclosure wire 86. The ends 88, 90 of the closure wire are then wrappedover the rolled portion to prevent that portion from unrolling (notshown in the drawings).

Referring now to FIGS. 5 and 6, incubation conditions depend on culturesample type. An incubator 58 is pictorially shown in FIG. 5. A pouch 10constructed according to the above description is shown sitting on ahorizontal shelf 60 within the incubator 58. The pouch 10 is shownholding a medical syringe and needle 62. In this case, the syringe andneedle 62 are being cultured to determine whether it is contaminated.

After incubation, it is a simple matter to inspect the cultured sampleand conduct any additional testing required (see 58 in FIG. 3).

When using pouches like the ones disclosed here, it is possible to storeprefilled and presterilized pouches more than twelve months prior toshipment or use. The following examples are provided to furtherillustrate specific embodiments of the method:

EXAMPLE 1

A sterile, flexible, stand-up pouch is prefilled with 225 ml of sterileBuffered Peptone Water for growing Salmonella bacteria as part of methodfor detecting this organism in a 25 gram food sample. The prefilledpouch is opened, 25 grams of sample is added, and the pouch is resealedby closing the ziplock. If a particulate sample is being analyzed, thepouch is placed in a blender with reciprocating paddles and blended for1 minute. The pouch is then incubated at 350 C. for 24 hours (+/2hours). Following incubation, aliquots of the growth medium are removedand analyzed using a rapid methods procedure such as an enzymeimmunoassay, a gene probe detection method, or by a traditional pureculture method as described in the Bacteriological Analytical Manual(FDA, 8th Ed.).

By starting with a different liquid culture medium in the stand-uppouch, 25 gram samples can be assayed for different pathogenic bacteriasuch as E. coli 0157, Staphylococcus aureus, Listeria spp., andCampylobacter spp.

EXAMPLE 2

A sterile, flexible, plastic bag is prefilled with 375 ml of sterileBuffered Peptone Water for growing Salmonella bacteria as part of amethod for detecting this organism in a 375 gram composite food sample.The prefilled bag is opened, 375 grams of sample added, and resealed byrolling down the bag at the opening and sealing with a clip. If aparticulate sample is being analyzed, the prefilled bag is placed in ablender with reciprocating paddles and blended for 1 minute by batteringthe sides of the bag with the paddles. If the prefilled bag is anordinary bag without the stand up, gusseted feature, then it is placedin a wire rack for holding the bag in a stand-up position and incubatedat 35° C. for 24 hours (+/−2 hours). Following incubation, aliquots ofthe growth medium are removed and analyzed using a rapid methodsprocedure such as an enzyme immunoassay, a gene probe detection method,or by a traditional pure culture method as described in manuals such asthe Bacteriological Analytical Manual (FDA, 8th Ed.).

EXAMPLE 3

Sterility testing of aseptically packaged foods, pharmaceutical products(e.g., injectable solutions, vaccines), and sterile medical devices(such as needles, catheters, etc.) can be accomplished by adding asample into sterility testing media such as sterile Tryp Soy Broth orsterile Thioglycollate Broth in a flexible stand-up pouch. With thefood, pharmaceutical, and cosmetic samples, a 10 gram sample is added to100 ml of Tryp Soy Broth and/or 100 ml of Thioglycollate Broth. If thesample is particulate in nature, the pouch is placed in a reciprocatingpaddle blender for 1 minute to homogenize the sample. The pouch withsample is incubated at 35° C. for 5-7 days and observed for cloudinessin the medium. Objects such as catheters can be added directly into thepouch containing the sterility broths without blending beforeincubation. Objects that are soft and porous (e.g., gauze, sponges) maybenefit from blending with a reciprocating paddle machine.

EXAMPLE 4

Coliform testing of water and wastewater samples is accomplished byadding 100 ml of sample to a pouch containing a nutrient mediumsupplemented with the reagent o-nitrophenyl β-Dgalactopyranoside (ONPG).The medium may be made as a concentrate such that the addition of thewater brings the constituents up to the correct final concentrations.Alternatively, the medium may be supplied as a powder which isreconstituted to the correct final constituent concentrations with theaddition of water. The sample is incubated in the pouch at 35° C. for 24hours. If coliform bacteria are present, the colorless ONPG compound isconverted to a yellow color.

EXAMPLE 5

E. coli testing of water and wastewater samples is accomplished byadding 100 ml of sample to a pouch containing a nutrient medium (e.g.,lauryl sulfate broth) supplemented with the reagent4-methylumbelliferyl-β-D-glucuronide (MUG). The medium may be made as aconcentrate such that the addition of the water brings the constituentsup to the correct final concentrations. Alternatively, the medium may besupplied as a powder which is reconstituted to the correct finalconstituent concentrations with the addition of water. The sample isincubated in the pouch at 35° C. for 24 hours. If E. coli bacteria arepresent, the colorless MUG compound is converted to a fluorescent bluishcompound that is observed under long wave (365 mm) ultraviolet light.

EXAMPLE 6

Environmental surface samples, such as floors, drains, and equipment ina food company plant, are analyzed for the presence of microorganismssuch as E. coli, Listeria spp., and Salmonella. These samples arecollected using sterile swabs or sponges. Swabs or sponges can be addedto a flexible stand-up pouch containing 100 ml of lauryl sulfate brothwith MUG (for E. coli), UVM broth (for Listeria), or Buffered PeptoneWater (for Salmonelia). The broth with the swab or sponge is incubatedfor-18-24 hours at 35° C. After incubation, the liquid culture medium isobserved for the presence of bluish fluorescent material under long wave(354 mm) ultraviolet light for the E. coli test, or aliquots of thegrowth medium are removed and analyzed using a rapid methods proceduresuch as an enzyme immunoassay, a gene probe detection method, or by atraditional pure culture method as described in manuals such as theBacteriological Analytical Manual (FDA, 8th Ed.) for Listeria andSalmonella testing.

EXAMPLE 7

Fifty grams of food sample such as meat is added to a flexible, plasticbag which incorporates a plastic mesh bag and is prefilled with 450 mlof sterile Butterfield's Phosphate Buffer. With even distribution of thesample, this dilutes the meat 1:10. The pouch is placed into a machinewith reciprocating paddles and blended for 1 minute. A one ml aliquot isremoved from the pouch using a serological pipette by accessing thediluent on the opposing side of the mesh bag from the meat. Thisminimizes the possibility of particulates clogging the serologicalpipette. A quantitative analysis is performed using a pour or spreadplate procedure or using a most probable number (MPN) procedure.

With respect to Example 7 above, and referring to FIG. 7, referencenumeral 70 illustrates a mesh bag inserted into the gusseted bag. Thetop ends of the mesh bag extend above the surface of the culture medium,and the bottom of the mesh bag extends towards the bottom of thegusseted bag to a sufficient depth. As mentioned in the precedingparagraph, samples are then introduced into the mesh bag andhomogenized. The ,aliquot,, is a sample which is then removed from underthe mesh 70 and is cultured in a manner other than incubation. The“pour” or “spread plate” procedures described in the samples would befamiliar to a person skilled in the art.

Alternatively, the gusseted bag 10, mesh bag 70, culture medium 14, andsample can all be incubated together, and afterwards, an aliquot isremoved by a pipette to perform tests for pathogenic organisms.

Having described the invention, it is to-be understood that its scope isnot to be limited by the specific embodiments disclosed above. Whileseven examples are described, it is not intended that the invention belimited to the examples. In some cases, it may be desirable to usenon-gusseted pouches to practice the procedure described above.

The scope of the invention is to be limited only by the patent claimswhich follow, the interpretation of which is to be made in accordancewith the standard doctrines of patent claim interpretation. Claim termsare to be interpreted first in accordance with their plain and ordinarymeaning. Dictionary definitions will be suitable to explain claim termsunless expressly indicated otherwise in some cases, dictionaries setforth more than one definition for the same word. In such case,applicant intends that the broadest definition be applied. In somecases, the claims use terms or expressions like “presterilizing themedium and a flexible stand-up plastic bag,” followed by a separateclause which reads “prefilling the bag with the medium.” It isspecifically intended that this expression covers both instances wherethe two items are either sterilized separately or together at the sametime.

What is claimed is:
 1. A method for producing a ready-to-use liquidculture medium, the liquid medium having the capability of being storedfor a period of time and shipped to the place where the medium is to beused, the method comprising: adding a medium to a flexible, two-sided,thin film plastic bag, including prefilling the bag with the medium,followed by sealing the bag, and wherein, the bag has a gussetted basethat is shaped to enable the bag to stand alone in a verticalorientation when filled, and further, the bag has a closure wire forresealing the bag by rolling the upper portion of the bag about theclosure wire and wrapping ends of the closure wire over the rolledportion of the bags; sterilizing the bag and medium; and delivering thesealed bag to a laboratory.
 2. A method for producing a ready-to-useliquid culture medium, the liquid medium having the capability of beingstored for a period of time and shipped to the place where the medium isto be used, the method comprising: adding a medium to a flexible, thinfilm plastic bag, including prefilling the bag with the medium, followedby sealing the bag, wherein, the bag has a gusseted base that is shapedto enable the bag to stand alone in a vertical orientation when filled,and further, the bag has a reopenable zip-lock closure for sealing thebag; sterilizing the bag and medium; and delivering the sealed bag to alaboratory for use.
 3. A method for producing a ready-to-use liquidculture medium, the liquid medium having the capability of being storedfor a period of time and shipped to the place where the medium is to beused, the method comprising: adding a medium to a flexible, thin filmplastic bag, including prefilling the bag with the medium, followed bysealing the bag, wherein, the bag has a gusseted base that is shaped toenable the bag to stand alone in a vertical orientation when filled, andfurther, the bag has a resealable closure for sealing the bag;sterilizing the bag and medium; and delivering the sealed bag to alaboratory for use.
 4. A method for producing a ready-to-use liquidculture medium, the liquid medium having the capability of being storedfor a period of time and shipped to the place where the medium is to beused, the method comprising: adding a medium to a flexible, thin filmplastic bag, including prefilling the bag with the medium, followed bysealing the bag, wherein, the bag has a gusseted base that is shaped toenable the bag to stand alone in a vertical orientation when filled;sterilizing the bag and medium; and delivering the sealed bag to alaboratory for use.